Nucleic Acids occur in nature as chains of either ribonucleotides or deoxyribonucleotides, the individual nucleotides being linked to each other by phosphodiester bonds between the ribose and deoxyribose sugars which form, respectively, the backbones of ribonucleic nucleic acid (RNA) or deoxyribonucleic acid (DNA). Apart from their role in naturally occurring phenomena, DNA and RNA, particularly DNA and RNA oligonucleotides, are expected to play an increasingly important role in medical diagnostic and therapeutic applications. For example, oligonucleotides have been shown to be useful in a variety of "probe" assays for viral and bacterial diseases and for the detection of genetic abnormalities. In these assays, the "probe" is typically an oligonucleotide selected to complement an RNA or DNA sequence which is unique to the organism or genetic defect to be detected.
It has also been observed that oligonucleotides which are complementary to messenger RNA (antisense oligonucleotides) can be introduced to a cell and arrest the translation of the mRNA. This arrest is believed to result from the hybridization of the antisense oligonucleotide to the mRNA. See, for example, Stephenson, et al , Proc. Natl. Acad. Sci., USA, 75, 285 (1978) and Zamecnick, et al., Proc. Natl. Acad. Sci.. USA, 75, 280 (1978).
The ability of antisense oligonucleotides to inhibit or prevent mRNA translation suggests their application in antiviral therapy. A virus infecting a cell reproduces its genetic information by using the biological machinery of the infected cell. Transcription and translation of that information by the cellular ribosomes are essential to viral reproduction. Thus, if expression of the viral gene can be interrupted, the virus cannot replicate or may replicate at such a slow rate as to give the host's immune system a better opportunity to combat the infection.
It has been proposed to use oligonucleotides in viral therapy by designing an oligonucleotide with a nucleotide sequence complementary to a sequence of virally expressed mRNA which must be translated if viral replication is to be successful. Introduction of the antisense oligonucleotide to the cell permits it to hybridize with and prevent, or at least inhibit, this essential translation.
Conventional phosphodiester antisense oligonucleotides have been reported to exhibit significant shortcomings as antisense oligonucleotides. One limitation is that they are highly subject to degradation by nucleases, enzymes which breakdown nucleic acids to permit recycling of the nucleotides. In addition, most cells are negatively charged. As a result, a phosphodiester oligonucleotide does not readily penetrate the cell membrane because of the density of negative charge in the backbone of the oligonucleotide.
It has been proposed to modify oligonucleotides to overcome these shortcomings. One such proposal has been to use non-polar analogs of conventional phosphodiester oligonucleotides. Such analogs retain the ability to hybridize with a complementary sequence and would be expected to enter the cell more readily and be less resistive to nuclease degradation. Promising results have been obtained with methyl phosphonate analogs. See Agris et al., Biochemistry 25, 1228 (1986). More recently thiophosphorate analogs, i.e., nucleic acids in which one of the non-bridging oxygen atoms in each inter-nucleotide linkage has been replaced by a sulfur atom, have also been shown to possess the ability to block mRNA translation. In at least one case, inhibition of expression of the chloramphenicol acetyltransferase gene, a thiophosphorate analog has been shown to be superior to the methyl phosphonate analog which in turn was shown to be more effective than the unmodified phosphodiester oligonucleotide. Inhibition of HIV virus replication by a thiophosphorate analog has also been demonstrated. See Matsukara et al, Proc. Natl. Acad. Sci., USA, 84, 1 (1987).
Thiophosphorate analogs of oligonucleotide probes are also useful as replacements for conventional probes in diagnostic applications as described above, and in other applications of oligonucleotides. However, only a few techniques have been reported for the synthesis of phosphorothioate analogs of nucleic acids, all of them cumbersome and not well adapted for use with currently available automated nucleic acid synthesizers.
One reported synthetic technique, for example, uses presynthesized nucleotide dimers. The synthesis of the full array of sixteen dimers necessary for the procedure is both laborious and expensive.
A more preferred procedure would permit use of the highly reactive, commercially available nucleoside-phosphoramidite monomers currently employed with nucleic acid synthesizers. Such monomers are actually used in processes for preparing phosphorothioate analogs. However, the sulfurization of phosphorous in the phosphite intermediate has been very troublesome. For example, elemental sulfur in pyridine at room temperature requires up to 16 hours to produce internucleotide phosphorothioate triester 12. (P. S. Nelson, et al., J. Org. Chem., 49, 2316 (1984); P. M. S. Burgers, et al., Tet Lett., 40, 3835 (1978)). A similar procedure using elemental sulfur, pyridine and carbon disulfide permitted sulfurization to be done at room temperature within 2 hours. B. A. Connolly, et al., Biochem., 23, 3483 (1984). The triester is convertible to the phosphorothioate by base catalyzed removal of substituent "R."
Carrying out the sulfurization at 60.degree. C. in 2,6-lutidine requires 15 minutes during automated, solid-phase synthesis of phosphothioates from Compound 11. W. J. Stec et al., J. Am. Chem. Soc., 106, 6077 (1984). However, most automated synthesizers do not have provisions for heating the column required for performing sulfurization at elevated temperature and vaporization of the solvent at 60.degree. C. would be expected to form bubbles in delivery lines which would reduce flow rates and even cause synthesis failures. In addition, even a fifteen minute reaction time for sulfurization after the addition of each nucleotide makes the procedure far from optimal.
Accordingly, there has gone unmet a need for a process for the preparation of phosphothioate oligonucleotide analogs that is rapid and that lends itself to use on conventional nucleic acid synthesizers.